Aspects of innate immunity in Sjögren's syndrome.

Abstract

Previously, a dominant role of the adaptive immune system in the pathogenesis of Sjögren's syndrome was suspected. Recent advances, however, have revealed a major role of the type I IFN pathway, documented by an increased circulating type I IFN activity and an IFN 'signature' in peripheral blood mononuclear cells and minor salivary gland biopsies from the patients. Polymorphisms in the genes IRF5 and STAT4 leading to increased IFN activation are associated with disease susceptibility. In the pathogenesis of Sjögren's syndrome, the activation of salivary gland epithelial cells appears to be the initial event. Once intrinsically activated, they express costimulatory and Toll-like receptors (TLRs) and MHC class I and II molecules, can present autoantigens and produce proinflammatory cytokines. The subsequent activation of plasmacytoid dendritic cells induces the production of high levels of proinflammatory cytokines in individuals with the risk alleles of the susceptibility genes IRF5 and STAT4. Under the influence of the high IFN concentration in the glands and through TLR ligation, B-cell activating factor is produced by epithelial cells and, together with autoantigen presentation on salivary gland epithelial cells, stimulates the adaptive immune system. In view of the central role of IFNalpha in at least the initiation of the pathogenesis of Sjögren's syndrome, blockade of this cytokine may be a rational therapeutic approach.

Full text

Page 1

Introduction
Sjögren’s syndrome (SS) is an autoimmune disorder
aff ecting the lachrymal and salivary glands and leads to
dry eyes and dry mouth. Due to the presence of lympho-
cytic infi ltrates in the glands and the presence of auto-
antibodies (rheumatoid factors and antibodies against
SS-A, SS-B, muscarinic receptors and alpha-fodrin), SS
has been regarded as a disorder that is caused by aberra-
tions in the adaptive immune system. Recent evidence
reviewed here, however, points to a major contribution of
the innate immune system, at least in the initiation of the
pathogenesis of SS.
Genetic susceptibility factors of Sjögren’s
syndrome
Th e etiology of SS is still unclear. Since there is a familial
aggregation of primary SS, however, genetic susceptibility
factors have been suspected for a long time. Initially,
HLA haplotypes were shown to be associated with
primary SS. Later on, however, it became clear that they
are primarily associated only with the subset of patients
with SS-A (HLA-DRB1*15) or SS-A and SS-B antibodies
(HLA-DRB1*03), but not with all subsets of SS. Currently,
genome-wide association studies are being performed to
identify the susceptibility genes of SS. So far, the genes
IRF5 (Interferon regulatory factor-5) and STAT4 (Signal
transducer and activator of transcription 4) have been
convincingly identifi ed and replicated in several studies
as susceptibility factors of primary SS independent of the
presence of autoantibodies.
Interferon regulatory factor-5
IRF5 is a transcription factor that mediates virus- and
IFN-induced signaling pathways. Infection of cells with
various viruses can activate Toll-like receptors (TLRs)
and, further downstream, IRF5 to induce IFNalpha and
the transcription of numerous infl ammatory proteins [1].
IRF5-/- mice are highly vulnerable to both DNA and RNA
viruses and infection of them was accompanied by low
IFNalpha concentrations in the sera [2].
Th ree studies have confi rmed an association between a
polymorphism in the IRF5 gene and primary SS. In a
French study [3], the IRF5 SNP rs2004640 GT or TT
genotype was identifi ed in 87% of primary SS patients but
in only 77% of controls (odds ratio (OR) 1.93). Th e IRF5
rs2004640 T allele was found on 59% of chromosomes
from primary SS patients compared with 52% of chromo-
somes from controls (OR 1.36). In a study of patients
from Sweden and Norway [4], a 5-bp CGGGG indel in
the promoter of IRF5 that is adjacent to rs2004640 was
associated with primary SS (OR 1.63). In another French
study [5], the 5-bp CGGGG indel in the promoter of the
IRF5 allele was confi rmed to transmit an increased risk of
primary SS in two cohorts (odds ratio 2.0).
Abstract
Previously, a dominant role of the adaptive immune
system in the pathogenesis of Sjögren’s syndrome was
suspected. Recent advances, however, have revealed
a major role of the type I IFN pathway, documented
by an increased circulating type I IFN activity and an
IFN ‘signature’ in peripheral blood mononuclear cells
and minor salivary gland biopsies from the patients.
Polymorphisms in the genes IRF5 and STAT4 leading
to increased IFN activation are associated with
disease susceptibility. In the pathogenesis of Sjögren’s
syndrome, the activation of salivary gland epithelial
cells appears to be the initial event. Once intrinsically
activated, they express costimulatory and Toll-like
receptors (TLRs) and MHC class I and II molecules, can
present autoantigens and produce proinfl ammatory
cytokines. The subsequent activation of plasmacytoid
dendritic cells induces the production of high levels
of proinfl ammatory cytokines in individuals with the
risk alleles of the susceptibility genes IRF5 and STAT4.
Under the infl uence of the high IFN concentration in
the glands and through TLR ligation, B-cell activating
factor is produced by epithelial cells and, together with
autoantigen presentation on salivary gland epithelial
cells, stimulates the adaptive immune system. In view
of the central role of IFNalpha in at least the initiation
of the pathogenesis of Sjögren’s syndrome, blockade of
this cytokine may be a rational therapeutic approach.
© 2010 BioMed Central Ltd
Aspects of innate immunity in Sjögren’s syndrome
Hui Zhi Low and Torsten Witte*
REVIEW
*Correspondence: witte.torsten@mh-hannover.de
Clinic for Immunology and Rheumatology, Medical School Hannover,
Carl-Neuberg-Str. 1, 30625 Hannover, Germany
Low and Witte Arthritis Research & Therapy 2011, 13:218
http://arthritis-research.com/content/13/3/218
© 2011 BioMed Central Ltd

Page 2

Th e CGGGG indel polymorphism of IRF5 is also
associated with other autoimmune disorders, such as sys-
temic lupus erythematosus (SLE) [6,7], rheumatoid
arthritis [8], and infl ammatory bowel disease [9], suggest-
ing common pathways in the induction of autoimmune
disorders.
In functional studies, the presence of the risk allele was
correlated with a high level of IRF5 mRNA in both
peripheral blood mononuclear cells (PBMCs) and salivary
gland epithelial cells (SGECs) and with increased levels of
mRNA transcripts of the IFN-induced genes MX1 and
IFITM1 [5]. As further evidence for the functional impact
of the risk allele, increased expression of IRF5 mRNA
from a promoter containing that allele was found using a
minigene reporter. Increased expression of IRF5 protein
was also observed in PBMCs from SLE patients carrying
the risk allele of the CGGGG indel [6].
Signal transducer and activator of transcription 4
Th e STAT4 transcription factor plays a key role in signal-
ing via the IFNalpha receptor by being activated and
translocated to the nucleus after receptor ligation [10].
Besides its role in type I IFN signaling, STAT4 is also
induced by IL-12 and IL-23 production by macrophages
and dendritic cells, and is responsible for the IL-12-
dependent activation of natural killer (NK) cells, polariza-
tion of naïve CD4+ T cells to IFNgamma-producing Th 1
cells and the IL-23-dependent expansion of Th 17 cells.
Th us, STAT4 has many stimulatory eff ects on the
immune system and may contribute to autoimmune
responses by aff ecting the functions of both innate and
adaptive immune cells.
Association studies of SNPs in the STAT4 gene revealed
that the T allele of rs7574865 was more common in
primary SS patients (on 29.6% of chromosomes) than in
controls (on 22.3% of chromosomes) [11]. Th e fi ndings
were confi rmed in a cohort from Colombia and Germany,
in which the T allele was again associated with primary
SS (OR 1.40) [12]. Th e C allele of the SNP rs7582694 of
the STAT4 gene, which is in complete linkage disequili-
brium with SNP rs7574865, was associated with primary
SS in a French cohort (OR 1.57) [13].
Polymorphism of STAT4 has also been associated with
other autoimmune disorders. Th e haplotype marked by
the SNP rs7574865 was more common in SLE patients of
European ancestry (OR 1.55) [14], in SLE patients from
the US and Sweden (OR 1.57) [15] and in a Chinese Han
population (OR 1.51) [16].
Th e T allele of SNP rs7574865 was also found to be
asso ciated with rheumatoid arthritis [14,17], with ORs
comparable to those observed in SLE, and, in a Spanish
study, with susceptibility to limited cutaneous systemic
sclerosis (OR 1.61), but not with diff use cutaneous sys-
temic sclerosis [18]. Th ese data have been confi rmed in a
combined meta-analysis of the Spanish cohort and fi ve
independent cohorts of European ancestry [18].
Th ere was no signifi cant association of any of the
STAT4 genotypes with mRNA levels of STAT4α and
STAT4β among 30 primary SS patients [13]. Th ere was,
however, a weak correlation of STAT4 rs7574865 and
rs7582694 polymorphisms, which are in complete linkage
disequilibrium, with STAT4α mRNA levels in PBMCs
from healthy donors [19]. In addition, the presence of the
SNP correlated with increased expression of the risk
allele of STAT4β in primary cells of mesenchymal origin
(osteoblasts) [20].
Th ose patients who carry all of the IRF5 and STAT4 risk
alleles have an increased risk (OR = 6.78) for primary SS
[4]. Th e association of both STAT4 and IRF5 polymor-
phisms with many autoimmune diseases that are type 1
IFN driven suggests that STAT4 and IRF5 can contribute
to a general loss of tolerance and that IFN is also a major
player in the induction of primary SS.
TREX-1
Recently, mutations in the TREX-1 gene (which encodes
the most abundant 3’-5’ DNA exonuclease in cells [21])
have been found to be tightly linked with the develop-
ment of autoimmune diseases, including primary SS.
Loss of function mutations of the human TREX-1 gene
cause Aicardi-Goutieres syndrome [22], which presents
as severe encephalitis in infants, a disorder resembling a
congenitally acquired viral infection. Patients with
Aicardi-Goutieres syndrome have elevated levels of type
I IFN in the cerebrospinal fl uid. Mutations in the TREX-1
gene have also been associated with monogenic chilblain
lupus [23] and later with SLE [24]. Most of the cases we
contributed to the latter analysis were patients who had
suff ered from SLE and secondary SS, and subsequent
careful exami na tion of relatives of these index subjects
revealed that several family members carrying the
TREX-1 mutations also suff ered from primary SS (un-
pub lished observa tions).
It has been suggested that TREX-1 mutations result in
defective clearance of intracellular DNA, in particular
from endogenous retroelements, which in the absence of
functional TREX-1 induces the production of type I IFN
and thus autoimmunity [25]. Trex-1 knockout mice die
from infl ammatory myocarditis at an early age [26], and
in Trex-1-defi cient mice single-stranded DNA fragments
derived from endogenous retroelements have been
shown to accumulate in the heart and induce myocarditis
[25]. Th e accumulation of single-stranded DNA in the
absence of Trex-1 induces the production of type I IFN
and a double knockout of Trex-1 and the type I IFN
receptor protected mice from developing the myocarditis
observed in the Trex-1 single knockout [23], suggesting
that IFN has a crucial role in this model of autoimmunity.
Low and Witte Arthritis Research & Therapy 2011, 13:218
http://arthritis-research.com/content/13/3/218
Page 2 of 7

Page 3

Taken together, the observations from recent genetic
studies point to a major infl uence of the type I IFN
pathway, and thus innate immunity, on the pathogenesis
of primary SS.
IFN signature in primary Sjögren’s syndrome
As described above, the function of the susceptibility
genes of SS suggest an important role of type I IFN in its
pathogenesis. Indeed, two transcriptome studies docu-
mented an IFN signature in the salivary glands of patients
with primary SS [27,28]. Using a complementary DNA
microarray to compare gene expression profi les in minor
salivary glands obtained from ten patients with primary
SS and ten control subjects, there was an upregulation of
numerous type I IFN-induced genes in primary SS [27].
Furthermore, global gene expression profi ling of minor
salivary gland cells revealed that the expression of 23
genes in the IFN pathway, including two encoding TLRs
(TLR8 and TLR9), was signifi cantly diff erent between
patients with primary SS and controls [28]. In addition,
mRNA obtained from peripheral blood of patients with
primary SS revealed a pattern of overexpression of IFN-
induced genes [29]. Approximately 50% of the over-
expressed genes in PBMCs from the peripheral blood of
patients with primary SS were found to be IFN inducible.
It has been diffi cult to detect elevated concentrations of
IFN itself in the blood of patients with SS, most likely due
to technical challenges with the commercially available
ELISA kits. However, it was recently shown that serum
and plasma from patients with primary SS can induce
IFN-regulated genes in PBMCs from control donors [30].
In addition, higher concentrations of IFNalpha and
IFNbeta in the serum of patients with primary SS were
found when cell reporter assays were used, which are
more sensitive than ELISAs [31].
Plasmacytoid dendritic cells (PDCs) are the most potent
producers of IFNalpha, producing up to 1,000 times more
type I IFN than other cells. Circulating PDCs express
higher levels of the activation marker CD40 in patients
with primary SS. Th e number of PDCs in the blood of
primary SS patients is reduced, but in immuno histo-
chemistry studies these IFN-producing cells were detected
in the salivary glands of all patients with primary SS but
not in controls [28]. Th ese results imply an infl ux of PDCs
from the blood into the infl amed tissues in primary SS.
Salivary gland epithelial cells
Immunohistochemistry revealed that the lymphocytic
infi ltrates in SS form around epithelial structures of
aff ected organs - for example, around the glandular epi-
thelium of the exocrine glands [32]. Th erefore, the term
‘autoimmune epithelitis’ was suggested for primary SS
[33] and research has focused on the role of epithelial
cells such as the SGECs.
Evidence for the activation of SGECs has been provided
by immunohistochemical analyses showing that they
express MHC class I and II molecules, the costimulatory
molecules CD80 and CD86, the adhesion receptors
intercellular adhesion molecule (ICAM)-1 and vascular
cell adhesion molecule, and the local production of
various chemokines and cytokines (IL1, IL6, TNF family
member B cell-activating factor (BAFF)). In addition,
conjunctival epithelial cells have been found to present
the autoantigen SS-B (La) [34]. SGECs may thus promote
the formation of lymphoid follicles by attracting and
activating both B and T cells.
Th ese fi ndings were able to be replicated in vitro after
techniques had been developed for long-term culture of
SGECs. Cultured SGECs produce high amounts of BAFF
and express several TLRs [35]. In addition, SGECs have
been found to be prone to apoptosis. Th ey produce
exosomes, which are of endosomal origin and derive
from the fusion of endosomes/lysosomes with the plasma
membrane. Exosomes contain various proteins, including
MHC class I and II and costimulatory molecules, cyto-
skeletal proteins and chaperones, and play a role in the
exchange of cellular material and in the transfer of
antigens to dendritic cells. SGECs have been shown to
contain SS-A and SS-B [36], and therefore may initiate
the typical autoantibody response in primary SS.
It is not clear yet what factors are responsible for the
activation of SGECs in primary SS. Intriguingly, the signs
of activation of SGECs, such as upregulation of MHC
class I molecules, costimulatory molecules, TLRs and
BAFF, remain stable even after long-term culture,
demonstrating that these cells are intrinsically activated.
Whether or not the activating stimulus is a virus remains
unknown so far. SGECs appear to be the initially
activated cells in the pathogenesis of SS and other cell
types, such as PDCs, or components of the adaptive
immune system are activated subsequently via the
presentation of autoantigens or transfer of exosomes.
Pattern recognition receptors
Th e cellular part of the immune system consists of natural
killer cells, monocytes, macrophages, granulocytes,
dendritic cells and mast cells. Th e innate immune system
responds to antigens in an HLA class II-independent
manner. According to the danger model [37], an infl am-
matory response is initiated by conserved molecular
patterns that may be associated with both foreign
antigens and cellular components released by damaged
cells. In order to recognize the molecular patterns, cells
of the innate immune system express receptors able to
detect highly conserved pathogen-associated molecular
patterns (PAMPs), endogenous components released
from damaged cells (danger associated molecular patterns
(DAMPs)), also referred to as alarmins [38], and the loss
Low and Witte Arthritis Research & Therapy 2011, 13:218
http://arthritis-research.com/content/13/3/218
Page 3 of 7

Page 4

of MHC class I molecules. Th e receptors recognizing
DAMPs and PAMPs are termed pattern recognition
receptors (PRRs). So far, several PRRs have been
characterized, in particular TLRs, retinoic acid inducible
gene (RIG) I-like receptors and NOD-like receptors.
TLRs primarily bind to PAMPs in the extracellular space
and in endosomes: for example, TLR3 to viral double-
stranded RNA, TLR4 to lipopolysac charide, TLR7 to
single-stranded RNA and TLR9 to unmethylated DNA.
RIG-1 is an intracellular receptor that binds to nucleic
acids, and NOD1 and NOD2 are activated by bacterial
peptidoglycans.
According to the danger model, tissue stress (for
example, cell necrosis, hypoxia, low pH), and not only
exogenous pathogens, can produce danger signals. Th e
activation of antigen-presenting cells via PRRs leads to
the upregulation of MHC class I and II and costimulatory
molecules and to the secretion of proinfl ammatory
cytokines, such as type I IFN, by PDCs. Th ese factors
activate the adaptive immune system, which in turn may
produce autoantibodies. In SS, antibodies against SS-A,
which is complexed to hYRNA, form immune complexes
that can bind and stimulate TLRs. Th us, a vicious cycle
may be initiated in which the innate and adaptive
immune systems stimulate each other to give a perpetuat-
ing autoimmune response.
Genetic studies on the role of PRRs have revealed that
SNPs of NOD2 are associated with Crohn’s disease
[39,40] and Blau syndrome [41]. With regard to SS, there
is so far evidence for a role of TLRs expressed by SGECs,
which does not exclude a major role for other PRRs.
Role of Toll-like receptors in primary Sjögren’s
syndrome
SGECs express TLR3 and other TLRs. In comparison to
other cells types, TLR3 is expressed at particularly high
levels. In addition, RT-PCR analysis and functional
studies revealed transcriptional activation of TLR2, TLR3
and TLR4 in cultured SGECs of patients with primary SS
[35]. TLR3 binds double-stranded RNA of viral origin
and its synthetic analogue poly(I:C). TLR3 ligation
induces the production of proinfl ammatory cytokines
and upregulates BAFF production in SGECs.
Th e role of TLRs in the pathogenesis of SS has also
been addressed by studies in mice. Th e injection of
poly(I:C), a TLR3 agonist, stimulates the production of
type I IFN. Th e treatment rapidly induces a temporal
hypofunction of the salivary glands of most mice strains,
which recover after termination of the poly(I:C) treat-
ment. Mice defi cient in IFNalpha-receptor1 are partially
protected. In NZB/W F1 mice, however, which are prone
to the development of a lupus-like disease, TLR3 stimu-
lation induces severe sialadenitis [42]. Th e loss of saliva
production precedes lymphocyte infi ltration [42]. Four
months after the discontinuation of the innate immunity
stimulation, a lymphocytic infi ltrate developed with
forma tion of lymphoid aggregates in the salivary glands.
Th is animal model of primary SS illustrates the initial
sequential activation of innate immunity and the
subsequent activation of ada ptive immunity.
Stimulation of the adaptive immune system by
components of innate immunity
Type I and II IFNs are the main inducers of the
production of BAFF. Transgenic mice that overexpress
BAFF develop polyarthritis and hallmarks of both SLE
and SS, including infi ltrates in the salivary glands and
reduced saliva production [43]. Th e concentration of
BAFF was found to be increased in the sera of patients
with active SS [44], as well as in the salivary glands [45]
and the saliva. Increased BAFF production was detected
in T cells and monocytes as well as in salivary gland duct
cells. Epithelial cells, therefore, are not only a target for
the autoimmune response in primary SS, but also
important in perpetuating the disease since they can
present autoantigens and produce proinfl ammatory
cytokines, including BAFF.
Possible triggers of the IFN signature in primary
Sjögren’s syndrome
So far, the initial inducer of IFN overproduction and the
pathogenesis of SS has remained unclear. Th e type I IFN
signature would be well in line with a viral trigger of the
disease. For example, chronic sialadenitis is associated
with hepatitis C virus and HIV infection. Epstein-Barr
virus, retroviruses, enteroviruses and coxsackievirus have
been suggested to induce SS [46], even though there is
still no defi nitive proof of their contribution to the
disease.
O n the other hand, the female predominance of SS
suggests a role of hormones in its pathogenesis. In parti-
cular, a role for estrogen deprivation has been suspected,
as the disease often starts after menopause. Estrogen-
defi cient mice develop a disease similar to primary SS
[47]. Estrogen defi ciency induces aberrant class II MHC
expression in exocrine glands via interactions between
epithelial cells and PDCs. Th e expression of MHC class II
molecules is increased in the exocrine glands of
ovariectomized C57BL/6 (B6) mice compared to control
B6 mice. Th e salivary gland dendritic cells adjacent to the
apoptotic epithelial cells become activated. Estrogen
defi ciency also induces the overexpression of the trans-
cription factor retinoblastoma-associated protein 48
(RbAP48). Mice with transgenic overexpression of RbAp48
develop autoimmune exocrinopathy resembling SS, with
ocular and oral dryness, a lymphocytic infi ltrate in the
salivary and lachrymal glands, and production of
autoantibodies typical for SS (anti-SS-A, anti-SS-B, and
Low and Witte Arthritis Research & Therapy 2011, 13:218
http://arthritis-research.com/content/13/3/218
Page 4 of 7

Page 5

anti-fodrin). R bAP48 overexpression leads to activation
and apoptosis of epithelial cells that express MHC class II
molecules and the costimulatory molecules CD80, CD86
and ICAM [48].
Conclusion: current model of the pathophysiology
of primary Sjögren’s syndrome
SS appears to be triggered by environmental factors such
as viral infection or hypoestrogenism. In patients carry-
ing susceptibility genes predisposing to enhanced reac tion
of the innate immune system via IFN pathway proteins,
epithelial cells become activated and may also produce
IFN and other cytokines (Figure 1). Subse quently,
autoantigen presentation by the epithelial cells and BAFF
overproduction induced by IFN stimulates B- and T-cell
activation. Finally, autoantibodies against SS-A/SS-B,
alpha-fodrin and muscarinic receptors develop as a sign
of the involvement of the adaptive immune system.
In conclusion, there is increasing evidence that innate
immunity, in particular the production of IFNs and the
activation of epithelial cells via TLRs, plays a major role
in the initiation of the pathophysiology of SS. Th ese
fi ndings are encouraging for future trials using anti-IFN
antibodies as new biologicals in the treatment of primary
SS.
Abbreviations
bp, base pair; BAFF, TNF family member B cell-activating factor; DAMP, danger
associated molecular pattern; ELISA, enzyme-linked immunosorbent assay;
ICAM, intercellular adhesion molecule; IFN, interferon; IL, interleukin; IRF,
interferon regulatory factor; MHC, major histocompatibility complex; OR, odds
ratio; PAMP, pathogen-associated molecular pattern; PBMC, peripheral blood
mononuclear cell; PDC, plasmacytoid dendritic cell; PRR, pattern recognition
receptor; RbAP, retinoblastoma-associated protein; SGEC, salivary gland
epithelial cell; SLE, systemic lupus erythematosus; SNP, single nucleotide
polymorphism; SS, Sjögren’s syndrome; STAT, signal transducer and activator of
transcription; TLR, Toll-like receptor; TNF, tumor necrosis factor.
Competing interests
The authors declare that they have no competing interests.
Figure 1. Current model of the initiation of the pathogenesis of Sjögren’s syndrome. (1) The disease is triggered by either a virus or
hypoestrogenism. (2) Salivary gland epithelial cells (SGECs) become activated and start to express MHC class II molecules. (3) The subsequent
activation of plasmacytoid dendritic cells (PDCs) induces a high production of proinfl ammatory cytokines, including IFNalpha, in individuals with
the risk alleles of the susceptibility genes IRF5 and STAT4. (4) Under the infl uence of the high IFN concentration in the glands, TNF family member
B cell-activating factor (BAFF) is produced and, together with the autoantigen presentation on SGECs, stimulates the adaptive immune system.
IFN-?
Triggers
Virus infection
Hypoestrogenism
1
2
3
MHCII
4
PDC
SGEC
salivary gland
epithelial cell
interferon-?
TLR-3/7/9
plasmacytoid DC
MHC II
interferon-?
receptor
STAT4
IRF5
Exosomes
Apoptosis/
Necrosis
Antigen
Release
MHCII
BAFF
Autoimmune Basis of Rheumatic Diseases
This article is part of a series on Sjögren’s syndrome, edited by Thomas
Dörner, which can be found online at http://arthritis-research.com/
series/Sjögrens
This series forms part of a special collection of reviews covering major
autoimmune rheumatic diseases, available at:
http://arthritis-research.com/series/abrd
Low and Witte Arthritis Research & Therapy 2011, 13:218
http://arthritis-research.com/content/13/3/218
Page 5 of 7